With current levels of research in the field of plant molecular genetics and functional genomics, plant transformation is likely to become an increasingly important tool for plant improvement. Limitations of current transformation procedures are numerous but one most important deficiency of currently used techniques is that they result in random insertions of target genes in host genomes, leading to uncontrolled delivery and unpredictable levels of transgene expression. As a result, existing methods require many independent transgenic plants to be generated and analyzed for several generations in order to find those with the desired level or pattern of expression. The vectors for such non-targeted transformation must necessarily contain full expression units, as the subsequent transformation to the same site is impossible, thus limiting engineering capability of the process. A number of different approaches have been investigated in an attempt to develop protocols for efficient targeting of DNA at specific sites in the genome. These efforts include:                (i) attempts to improve the process of homologous recombination (that relies on the endogenous cellular recombination machinery) by over-expressing some of the enzymes involved in recombination/repair;        (ii) attempts to decrease non-targeted recombination by down-regulating enzymes that contribute to non-specific recombination;        (iii) use of heterologous recombinases of microbial origin;        (iv) development of chimeraplasty for targeted DNA modification in plants.        
A brief description of these efforts is summarized below.
Homologous recombination occurs readily in bacteria and yeast, where it is used for gene replacement experiments. More recently it has been developed as a tool for gene replacement in mammals (Mansour et al, 1988, Nature, 336, 348-336; Thomas et al, 1986, Cell, 44, 419-428; Thomas et al, 1987, Cell, 51, 503-512), and the moss Physcomytrella patens (Schaefer & Zryd, 1997, Plant J., 11, 1195-1206). However, it is inefficient in plants. Targeted DNA modification by homologous recombination is accomplished by introducing into cells linear DNA molecules that share regions of homology with the target site. Homologous recombination occurs as a result of a repair mechanism induced by the double-strand breaks at the ends of the DNA fragment. Unfortunately, a competing repair mechanism called non-homologous end-joining (NHEJ) also takes place at a much higher frequency in many organisms and/or cell types, rendering selection of the desired site-targeted events difficult (Haber, 2000, Curr. Op. Cell. Biol., 12, 286-292; Haber, 2000, TIG, 16, 259-264; Mengiste & Paszkowski, 1999, Bio.l Chem., 380, 749-758). In higher plants only a few cases of successful targeted transformation by homologous recombination have been reported, and all were obtained with efficiencies of targeted events over non-targeted events in the range of 10−3 to 10−5 (Paszkowski et al., 1988, EMBO J., 7, 4021-4026; Lee et al., 1990, Plant Cell; 2, 415-425; Miao & Lam, 1995, Plant J., 7, 359-365; Offringa et al., 1990, EMBO J., 9, 3077-3084; Kempin et al., 1997, Nature, 389, 802-803). This means that the screening procedure will involve a very large number of plants and will be very costly in terms of time and money; in many cases this will be a futile effort.
Attempts to increase homologous recombination frequencies have been made. Investigators have over-expressed some of the enzymes involved in double-strand break repair. For example, over-expression of either the E. coli RecA (Reiss et al., 1996, Proc Natl Acad Sci USA., 93, 3094-3098) or the E. coli RuvC (Shalev et al., 1999, Proc Natl Acad Sci USA., 96, 7398-402) proteins in tobacco has been tried. However, this has only led to an increase of intrachromosomal homologous recombination (of approximately 10 fold). There was no increase of gene targeting (Reiss et al., 2000, Proc Natl Acad Sci USA., 97, 3358-3363.). Using another approach to increase homologous recombination, investigators have induced double-strand breaks at engineered sites of the genome using rare cutting endonucleases such as the yeast HO endonuclease (Chiurazzi et al, 1996, Plant Cell, 8, 2057-2066; Leung et al., 1997, Proc. Natl. Acad. Sci., 94, 6851-6856) or the yeast I-Sce I endonuclease (Puchta et al., 1996, Proc. Natl. Acad. Sci., 93, 5055-5060). Site targeted frequency of 2×10−3 to 18×10−3 was obtained using the I-Sce I endonuclease. Although an improvement, this is still inefficient. In addition, many of the targeted events contained unwanted mutations or occurred by homologous recombination at one end of the break only. Incidentally, there is an interesting recent publication describing a hyperrecombinogenic tobacco mutant demonstrating three orders of magnitude increase of mitotic recombination between homologous chromosomes, but the gene(s) involved has not been identified yet (Gorbunova et al., 2000, Plant J., 24, 601-611) and targeted recombination is not involved.
An alternative approach consists of decreasing the activity of enzymes (e.g. Ku70) involved in non-homologous end joining (U.S. Pat. No. 6,180,850) to increase the ratio of homologous/non-homologous recombination events. This approach has been far from being practically useful.
A recently developed approach called chimeraplasty consists of using DNA/RNA oligonucleotides to introduce single-nucleotide mutations in target genes. This approach is highly efficient in mammalian cells (Yoon et al., 1996, Proc. Natl. Acad. Sci. USA., 93, 2071-2076; Kren et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 10349-10354; Bartlett et al., 2000, Nature Biotech., 18, 615-622) with a success rate of more than 40%. Unfortunately, the efficiency is much lower in plants (Zhu et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 8768-8773; Beetham et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 8774-8778; Zhu et al., 2000, Nature Biotech., 18, 555-558; WO9925853) and reaches only a frequency of 10−5-10−7. A further severe drawback of using the chimeraplasty approach in plant systems is that it is limited to the introduction of single-nucleotide mutations and to the special case where the introduced mutation results in a selectable phenotype.
Another approach has been to use heterologous site-specific recombinases of microbial origin. When these recombinases are used, specific recombination sites have to be included on each side not only of the DNA sequence to be targeted, but also of the target site. So far, this has been a severely limiting condition which gives this approach little practical usefulness. Examples of such systems include the Cre-Lox system from bacteriophage P1 (Austin et al., 1981, Cell, 25, 729-736), the Flp-Frt system from Saccharomyces cerevisiae (Broach et al., 1982, Cell, 29, 227-234), the R-RS system from Zygosaccharomyces rouxii (Araki et al., 1985, J. Mol. Biol., 182, 191-203) and the integrase from the Streptomyces phage PhiC31 (Thorpe & Smith, 1998, Proc. Natl. Acad. Sci., 95, 5505-5510; Groth et al., 2000, Proc. Natl. Acad. Sci., 97, 5995-6000). Wild-type Lox sites (LoxP sites) consist of 13 bp inverted repeats flanking an 8 bp asymetrical core. The asymmetry of the core region confers directionality to the site. Recombination between LoxP sites is a reversible reaction that can lead to deletions, insertions, or translocations depending on the location and orientation of the Lox sites. In plants, the Cre-Lox system has been used to create deletions (Bayley et al, 1992, Plant Mol. Biol., 18, 353-361), inversions (Medberry et al., 1995, Nucl. Acids. Res., 23, 485-490), translocations (Qin et al., 1994, Proc. Natl. Aced. Sci., 91, 1706-1710); Vergunst et al, 2000, Chromosoma, 109, 287-297), insertion of a circular DNA into a plant chromosome (Albert et al., 1995, Plant J., 7, 649-659), interspecies translocation of a chromosome arm (Heather et al., 2000, Plant J., 23, 715-722), and removal of selection genes after transformation (Dale & Ow, 1991, Proc. Natl. Acad. Sc., 88, 10558-62; Zuo et al., 2001, Nat Biotechnol., 19, 157-161). One problem encountered when the Cre-Lox system (or a similar recombination system) is used for targeted transformation is that insertion of DNA can be followed by excision. In fact, because the insertion of DNA is a bimolecular reaction while excision requires recombination of sites on a single molecule, excision occurs at a much higher efficiency than insertion. A number of approaches have been devised to counter this problem including transient Cre expression, displacement of the Cre coding sequence by insertion leading to its inactivation, and the use of mutant sites (Albert et al., 1995, Plant J., 7, 649-659; Vergunst et al., 1998, Plant Mol. Biol., 38, 393-406; U.S. Pat. No. 6,187,994). Some site-specific recombinases such as the Streptomyces phage PhiC31 integrase should not suffer from the same problem, theoretically, as recombination events are irreversible (the reverse reaction is carried out by different enzymes) (Thorpe & Smith, 1998, Proc. Natl. Acad. Sci., 95, 5505-5510), but they are limited to animals), but the use of this recombination system in plant cells has not been confirmed yet. There are other flaws that render the site-specific recombination systems practically unattractive. First, one needs to engineer a landing or docking site in the recipient's genome, a procedure that is currently done by random insertion of recombination sites into a plant genome. This eliminates most of benefits of the site-specific integration. Second, the frequency of desired events is still very low, especially in economically important crops, thus limiting its use to tobacco and Arabidopsis. Expression of recombinant enzymes in plant cells leads to a toxicity problems, an issue that cannot be circumvented with commonly used systems such as Cre-lox or Flp-frt.
WO 99/25855 and corresponding intermediate U.S. Pat. No. 6,300,545 disclose a method of mobilizing viral replicons from an Agrobacterium-delivered T-DNA by site-specific recombination-mediated excision for obtaining a high copy number of a viral replicon in a plant cell. It is speculated that said high copy number is useful for site-targeted integration of DNA of interest into a plant chromosome using site-specific recombination. However, the disclosure does not contain information on how to test this speculation. The examples given in the disclosure do not relate to site-targeted integration. Moreover, the examples cannot provide cells having undergone site-targeted integration, but only plants showing signs of viral infection such as appearance of yellow spots and stripes at the base of new leaves indicative of the decay of the infected cells. Therefore, the teaching of these references is limited to the infection of cells leading to the destruction of the cell by the viral vector. The teaching of these references neither allows the determination as to whether or not integration into the nuclear genome has taken place, let alone the selection of successful site-targeted integration events. This is underlined by the fact that the references do not contain a disclosure of selection methods for recovering site-targeted transformants. Selection and recovery of transgenic progeny cells containing said DNA of interest site-specifically integrated into the nuclear genome is simply impossible based on the teaching of these references. Moreover, WO 99/25855 and U.S. Pat. No. 6,300,545 are silent on this problem. Further, these documents are silent on homologous recombination. Moreover, the method is limited to replicon delivery by way of Agrobacterium. 
Therefore, it is the problem of the invention to provide a process for targeted transformation of plants which is sufficiently efficient for practical purposes.
It is a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome that allows recovery of integration events, i.e. selection of cells having undergone recombination in the plant nuclear DNA.
It is a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome by homologous recombination.
It is therefore a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome by delivery methods independent from Agrobacterium-mediated methods.